In Streptococcus mitis (SM), an oral streptococcus generally recognized as low pathogenic species, the strains with plural cholesterol-dependent cytolysins (CDC) were identified. Therefore, we carried out the study on pathogenicity and epidemiology of SM strains including their comparative genome analysis. It was revealed that highly pathogenic SM (HPSM) strains with penicillin G-resistance (PR) and potent cytotoxicity against human cells by human-directed CDC were frequently isolated from the patients of pneumonia and Kawasaki disease. As the results of comparative genome analysis among Mitis group streptococci, it was shown that all loci of CDC genes were individually classified into several patterns and the genomic hybridization with related species probably occurred at the loci nearby CDC genes. Thus, it was suggested that HPSM occurred through further recombination within or among species after the first transmission of CDC genes, and PR mutations.